Monoclonal antibodies cross-reactive and cross-protective against P. aeruginosa serotypes

ABSTRACT

Cell lines have been produced that secrete human monclonal antibodies capable of binding to the lipopolysaccharide molecules of selected Pseudomonas aeruginosa IATS serotypes. Pharmaceutical compositions containing these antibodies, which can be in combination with other monoclonal antibodies, blood plasma fractions and antimicrobial agents, and the prophylactic and therapeutic use of such compositions in the management of infections are included. 
     Prior to filing of this patent application the continuous transformed human cell lines 1C1, 6D6, and 8H7 described herein were deposited in the American Type Culture Collection and given the designations CRL 8941, 9171, and. 9258, respectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of Ser. No. 6/931,179,filed Nov. 24, 1986, now abandoned which is a continuation-in-part ofapplication Ser. No. 06/807,394, filed Dec. 10, 1985, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the application of immunologicaltechniques to provide novel materials useful in diagnosing and treatingbacterial infections and, more particularly, to the production andapplication of human monoclonal antibodies that are capable ofrecognizing multiple serotypes of Pseudomonas aeruginosa.

BACKGROUND OF THE INVENTION

Gram-negative disease and its most serious complications, e.g.,bacteremia and endotoxemia, are the cause of significant morbidity andmortality in human patients. This is particularly true of thegram-negative organism Pseudomonas aeruginosa, which has beenincreasingly associated with bacterial infections, especially nosocomialinfections, over the last fifty years.

During the past few decades, antibiotics have been the therapy of choicefor control of gram-negative disease. The continued high morbidity andhigh mortality associated with gram-negative bacterial disease, however,is indicative of the limitations of antibiotic therapy particularly withrespect to P. aeruginosa. (See, for example, Andriole, V. G.,"Pseudomonas Bacteremia: Can Antibiotic Therapy Improve Survival?", J.Lab. Clin. Med. (1978) 94:196-199). This has prompted the search foralternative methods of prevention and treatment.

One method that has been considered is augmentation of the host's immunesystem by active or passive immunization. For instance, it has beenobserved that active immunization of humans or experimental animals withwhole cell bacterial vaccines or purified bacterial endotoxins from P.aeruginosa leads to the development of specific opsonic antibodiesdirected primarily against determinants on the repeating oligosaccharideunits of the lipopolysaccharide (LPS) molecules located on the outercell membrane of P. aeruginosa (see Pollack, M., Immunoglobulins:Characteristics and Uses of Intravenous Preparations, Alving, B. M. andFinlayson, J. S., eds, pp. 73-79, U.S. Department of Health and HumanServices, 1979). Such antibodies, whether actively engendered orpassively transferred, have been shown to be protective against thelethal effects of P. aeruginosa infection in a variety of animal models(Pollack, supra) and in some preliminary investigations with humans (seeYoung, L. S. and Pollack, M., Pseudomonas aeruginosa, Sabath, L., ed.,pp. 119-132, Hans Huber, 1980). Moreover, of particular importance tothe role of these antibodies in humans has been the finding in patientswith P. aeruginosa bacteremia of an association between survival andhigh acute serum titers of antibodies to the LPS molecules of theinfecting strain (see Pollack, M., and Young, L. S., J. Clin Invest.,63:276-286, (1979)).

The above reports suggest that immunotherapeutic approaches could beutilized to prevent and treat bacterial disease due to P. aeruginosa,such as by administering pooled human immune globulins that containantibodies against the infecting strain(s). Human immune globulins aredefined herein as that portion of fractionated human plasma that isenriched for antibodies, among which are represented specific antibodiesto strains of P. aeruginosa. Due to certain inherent limitations inusing human immune globulin components, this approach to treatment ofdisease due to P. aeruginosa remains under investigation (see, forexample, Collins, M. S. and Roby, R. E., Am. J. Med., 76(3A):168-174,(1984)), and as yet there are no commercial products available utilizingthese components.

One such limitation associated with immune globulin compositions is thatthey consist of pools of samples from a thousand or more donors, suchsamples having been preselected for the presence of particularanti-Pseudomonas antibodies. This pooling leads to an averaging ofindividual antibody titers which, at best, results in modest increasesin the resultant titer of the desired antibodies.

Another limitation is that the preselection process itself requiresexpensive, continuous screening of the donor pool to assure productconsistency. Despite these efforts, the immune globulin products canstill have considerable variability from batch to batch and amongproducts from different geographic regions.

Yet another such limitation inherent in immune globulin compositions isthat their use results in the coincident administration of largequantities of extraneous proteinaceous substances (which may includeviruses, such as those recently shown to be associated with AcquiredImmune Deficiency Syndrome, or AIDS), having the potential to causeadverse biologic effects. The combination of low titers of desiredantibodies and high content of extraneous substances may often limit, tosuboptimal levels, the amount of specific and thus beneficial immuneglobulin(s) administrable to the patient.

There exists in the literature a number of serotyping schema that areuseful for analyzing Pseudomonas aeruginosa infections. The schema areprimarily based on the heat-stable major somatic antigens of thisorganism (see Zierdt, C. H., in Glucose Nonfermenting Gram-NegativeBacteria in Clinical Microbiology, Gilardi, G. L., ed., CRC Press, pp.213-238 (1978)). The proliferation of the serogrouping schema has madeserological studies of P. aeruginosa rather difficult to compare, andthus the choice of any given system for the purpose of screeningsupernatants would seem somewhat arbitrary. The confusion between typingsystems was recently clarified by the creation of the InternationalAntigenic Typing Scheme (IATS) system which was proposed by theSubcommittee on Pseudomonadaceae of the International Committee onSystematic Bacteriology as the backbone for further serological study ofP. aeruginosa. This system, which provides for seventeen distinctserotypes, designated IATS Type 1, IATS Type 2, etc., encompasses allthe heat-stable major somatic antigens identified in previous systems.See Liu, P. V., Int. J. Syst. Bacteriol., 33:256-264, (1983), which isincorporated herein by reference.

In developing protective monoclonal antibodies that are cross-reactiveamong strains of P. aeruginosa, it is also advantageous to incorporate atyping scheme which is based on the protective antigens of P.aeruginosa. Such a scheme has been devised with the intention ofdeveloping a vaccine for clinical use and is described in detail inFisher, M. W. et al., J. Bacteriol., 98:835-836, (1969). This system,commonly referred to as the Fisher typing system, classifies themajority of known P. aeruginosa into seven types, designated Fisherimmunotype 1, Fisher immunotype 2, etc. Correlation between the IATS andFisher typing systems has been clarified (see Liu, P. V., et al., Supra)and is presented in Table I. As noted in Table I, there are nocorresponding Fisher immunotypes for certain IATS serotypes, althougheach Fisher immunotype does correspond to a certain IATS serotype. Forthe IATS and Fisher typing systems, the antigenic determinants relevantto both serotyping schemes are believed to reside on the surface LPSmolecules of P. aeruginosa (Liu, P. V. et al., supra; Hanessien, F., etal., Nature, 229:209-210 (1979)).

                  TABLE I                                                         ______________________________________                                        Comparison and Correlation of the IATS and Fisher                             Typing Schemes for Pseudomonas aeruginosa                                             IATS  Fisher                                                          ______________________________________                                                1     4                                                                       2     3                                                                       3     --                                                                      4     --                                                                      5     7                                                                       6     1                                                                       7     --                                                                      8     6                                                                       9     --                                                                      10    5                                                                       11    2                                                                       12    --                                                                      13    --                                                                      14    --                                                                      15    --                                                                      16    --                                                                      17    --                                                              ______________________________________                                    

In 1975 Kohler and Milstein reported their seminal discovery thatcertain mouse cell lines could be fused with mouse spleen cells tocreate hybridomas each of which which would secrete antibodies of asingle specificity, i.e., monoclonal antibodies (Kohler, G., andMilstein, C., Nature, 256:495-497 (1975)). With the advent of thistechnology it became possible, in some cases, to produce largequantities of exquisitely specific murine antibodies to a particulardeterminant or determinants on antigens. Such mouse monoclonalantibodies or compositions of such antibodies may, however, have majordisadvantages for use in humans, particularly in light of the findingthat mouse monoclonal antibodies when used in trial studies for thetreatment of certain human disease have often been observed to elicit animmune response that renders them noneffective (Levy, R. L. and Miller,R. A., Ann. Rev. Med., 34:107-116, (1983)).

Using hybridoma technology, Sadoff et al. have reported the productionof a mouse monoclonal antibody of the IgM class directed against anO-side chain determinant on the LPS molecules of a particular serotypeof P. aeruginosa (Abstracts of the 1982 Interscience Conference onAntimicrobial Agents and Chemotherapy, No. 253). They further reportedthat this murine antibody protected mice against a lethal challenge ofP. aeruginosa of the same serotype as the type to which the antibodieswere directed (i.e., the homologous serotype). Several subsequentarticles have detailed the development of mouse and human anti-P.aeruginosa LPS monoclonal antibodies of various specificities; forexample: Sawada, S., et al., J. Inf. Dis., 150:570-576, (1984).; Sadoff,J. , et al. , Antibiot. Chemother., 36:134-146, (1985); Hancock, R., etal., Infect. Immun. 37:166-171 (1982); Siadak, A. W. and Lostrom, M. E.,in Human Hybridomas and Monoclonal Antibodies, Engleman, E. G., et al.,eds., pp. 167-185, Plenum Publishing Corp. (1985) and Sawada, S. et al.,J. Inf. Dis., 152:965- 970, (1985). Production and immunotherapeuticapplication of anti-P. aeruginosa LPS serotype-specific human monoclonalantibodies are disclosed in pending U.S. patent application Ser. Nos.734,624 now U.S. Pat. No. 4,834,975 and 828,005, now abandoned which areincorporated herein by reference.

While certain advantages may exist for utilizing monoclonal antibodiesspecific for a single IATS serotype of P. aeruginosa in some situations,such as when infection can be traced to a single serotype, in many othersituations they would not be preferred. For example, in prophylactictreatments for potential infections in humans, it would be preferable toadminister a human antibody or antibodies protective against a pluralityof IATS serotypes. Similarly, in therapeutic applications where theserotype(s) of the infecting strain(s) is not known, it would bepreferable to administer a human antibody or antibodies effectiveagainst most, if not all, of the clinically important P. aeruginosa IATSserotypes. Whereas a combination of human monoclonal antibodies, eachspecific for a single IATS serotype of P. aeruginosa, theoreticallymight be formulated to protect against the various serotypes, such acomposition would be difficult to develop and, from a manufacturingstandpoint, uneconomical to produce.

Accordingly there exists a significant need for human anti-P. aeruginosamonoclonal antibodies capable of recognizing and providing protectionagainst multiple IATS serotypes of P. aeruginosa. Further, theseantibodies should be suitable for use as prophylactic and therapeutictreatments of P. aeruginosa infections. The present invention fulfillsthese needs.

SUMMARY OF THE INVENTION

Novel cell lines are provided which can produce human monoclonalantibodies capable of specifically reacting with the LPS molecules of aplurality of, but not all, IATS serotypes of P. aeruginosa. Theseantibodies have various reactivities with Fisher immunotypes, binding tozero, one, or a plurality of immunotypes. Additionally, a method isprovided for treating a human susceptible to infection or alreadyinfected with P. aeruginosa by administering a prophylactic ortherapeutic amount of a composition comprising at least one humanmonoclonal antibody or binding fragment thereof capable ofcross-reacting with P. aeruginosa IATS serotypes, the compositionpreferably also including a physiologically acceptable carrier. Thecomposition may also contain any one or more of the following:additional human monoclonal antibodies capable of reacting with P.aeruginosa flagella, exotoxin A, or with other serotype or immunotypedeterminants on the LPS of P. aeruginosa; a gamma globulin fraction fromhuman blood plasma; a gamma globulin fraction from human blood plasma,where the plasma is obtained from humans exhibiting elevated levels ofimmunoglobulins reactive with P. aeruginosa; and one or moreantimicrobial agents.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the present invention, novel cells capable ofproducing human monoclonal antibodies and compositions comprising suchantibodies are provided, such compositions being capable of selectivelyrecognizing at least a plurality of, and in some cases all, P.aeruginosa strains, where individual antibodies typically recognizemultiple IATS serotypes and zero, one or more Fisher immunotypes of P.aeruginosa. The subject cells have identifiable chromosomes in which thegerm-line DNA from them or precursor cells have been rearranged toencode an antibody having a binding site for an epitope common amongcertain P. aeruginosa serotypes. These human monoclonal antibodies canbe used in a wide variety of ways, including diagnosis and therapy(e.g., protective in vivo).

Typically, the cells of the present invention will be human transformedlymphocytes that produce protective human monoclonal antibodies toaccessible LPS molecules of P. aeruginosa. By "accessible" is meant thatthe LPS molecules are physically available in the environment of use fordirect interaction with the monoclonal antibodies. The monoclonalantibodies so provided are useful in the treatment or prophylaxis ofserious disease due to P. aeruginosa. The LPS molecules of the outermembrane of P. aeruginosa would be available for direct contact by theantibody molecules, thus facilitating complement-mediated lysis and/orphagocytosis of the organism. Furthermore, those LPS molecules that areshed from the outer membrane into the surrounding environment would alsobe free to interact directly with the antibody molecules and be clearedvia the reticuloendothelial system.

The preparation of monoclonal antibodies can be accomplished byimmortalizing the expression of nucleic acid sequences that code forantibodies specific for an epitope on the LPS molecules of multipleserotypes of P. aeruginosa. Typically the monoclonal antibodies areproduced by cell-driven Epstein-Barr virus (EBV) transformation ofB-lymphocyte cells obtained from human donors who have been exposed tothe appropriate serotype(s) of P. aeruginosa. The antibody secretingcell lines so produced are characterized as continuously growinglymphoblastoid cells that possess a diploid karyotype, are Epstein-Barrnuclear antigen positive, and secrete monoclonal antibody of either IgG,IgM, IgA, or IgD isotype, including various subtypes such as IgG1, IgG2,IgG3 and IgG4. The cell-driven transformation process itself isdescribed in detail in U.S. Pat. No. 4,464,465, which is incorporatedherein by reference. The monoclonal antibodies may be used intact, or asfragments, such as Fv, Fab, F(ab')₂, but usually intact, prepared inaccordance with procedures well-known in the art.

Alternatively, cell lines producing the antibodies could be produced bycell fusion between suitably drug-marked human myeloma, mouse myeloma,human-mouse heteromyeloma, or human lymphoblastoid cells with humanB-lymphocytes to yield human hybrid cell lines.

The cell lines of the present invention may find use other than for thedirect production of the human monoclonal antibodies. The cell lines maybe fused with other cells (such as suitably drug-marked human myeloma,mouse myeloma, human-mouse heteromyeloma, or human lymphoblastoidcells), to produce hybridomas, and thus provide for the transfer of thegenes encoding the monoclonal antibodies. Alternatively, the cell linesmay be used as a source of the chromosomes encoding the immunoglobulins,which may be isolated and transferred to cells by techniques other thanfusion. In addition, the genes encoding the monoclonal antibodies may beisolated and used in accordance with recombinant DNA techniques for theproduction of the specific immunoglobulin in a variety of hosts.Particularly, by preparing cDNA libraries from messenger RNA, a singlecDNA clone, coding for the immunoglobulin and free of introns, may beisolated and placed into suitable prokaryotic or eukaryotic expressionvectors and subsequently transformed into a host for ultimate bulkproduction.

The lymphoblastoid or hybrid cell lines may be cloned and screened inaccordance with conventional techniques, with the antibodies that arecapable of binding to the epitopes of different P. aeruginosa IATSserotypes and Fisher immunotypes detected in the cell supernatants. Byutilizing antibodies of the present invention as blocking antibodies inscreening, in accordance with procedures well-known to those skilled inthe art, additional monoclonal antibodies recognizing the same antigenicdeterminants or epitopes can be readily isolated.

In one aspect of the present invention, human monoclonal antibodies areprovided that are capable of specifically binding to thelipopolysaccharide determinants present on a plurality of, but not all,IATS serotypes of P. aeruginosa. These determinants may be present, forexample, on two or three IATS serotypes, and on zero, one, or at leasttwo Fisher immunotypes. Such antibodies may be protective in vivoagainst some or all of the recognized serotypes and immunotypes,typically two or more serotypes.

Moncclonal antibodies of the present invention can also find a widevariety of utilities in vitro. By way of example, the monoclonalantibodies can be utilized for typing, for isolating specific P.aeruginosa strains, for selectively removing P. aeruginosa cells in aheterogeneous mixture of cells, or the like.

For diagnostic purposes, the monoclonal antibodies may either be labeledor unlabeled. Typically, diagnostic assays entail detecting theformation of a complex through the binding of the monoclonal antibody tothe LPS of the P. aeruginosa organism. When unlabeled, the antibodiesfind use in agglutination assays. In addition, unlabeled antibodies canbe used in combination with other labeled antibodies (second antibodies)that are reactive with the monoclonal antibody, such as antibodiesspecific for immunoglobulin. Alternatively, the monoclonal antibodiescan be directly labeled. A wide variety of labels may be employed, suchas radionuclides, fluorescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, ligands (particularly haptens), etc.Numerous types of immunoassays are available, and by way of example,some include those described in U.S. Pat. Nos. 3,817,827; 3,850,752;3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876,all of which are incorporated herein by reference.

Commonly, the monoclonal antibodies of the present invention areutilized in enzyme immunoassays, where the subject antibodies, or secondantibodies from a different species, are conjugated to an enzyme. When asample containing P. aeruginosa of a certain serotype, such as humanblood or lysate thereof, is combined with the subject antibodies,binding occurs between the antibodies and those molecules exhibiting thedesired epitope. Such cells may then be separated from the unboundreagents, and a second antibody (labeled with an enzyme) added.Thereafter, the presence of the antibody-enzyme conjugate specificallybound to the cells is determined. Other conventional techniques wellknown to those skilled in the art may also be utilized.

Kits can also be supplied for use with the subject antibodies in thedetection of P. aeruginosa infection or for the presence of P.aeruginosa antigen. Thus, the subject monoclonal antibody composition ofthe present invention may be provided, usually in a lyophilized form,either alone or in conjunction with additional antibodies specific forother gram-negative bacteria. The antibodies, which may be conjugated toa label or unconjugated, are included in the kits with buffers, such asTris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins,e.g., bovine serum albumin, or the like. Generally, these materials willbe present in less than about 5% wt. based on the amount of activeantibody, and usually present in total amount of at least about 0.001%wt. based again on the antibody concentration. Frequently, it will bedesirable to include an inert extender or excipient to dilute the activeingredients, where the excipient may be present in from about 1 to 99%wt. of the total composition. Where a second antibody capable of bindingto the monoclonal antibody is employed, this will usually be present ina separate vial. The second antibody is typically conjugated to a labeland formulated in an analogous manner with the antibody formulationsdescribed above.

Pharmaceutical Formulations and Use

The monoclonal antibodies of this invention can also be incorporated ascomponents of pharmaceutical compositions containing a therapeutic orprophylactic amount of at least one of the monoclonal antibodies of thisinvention with a pharmaceutically effective carrier. A pharmaceuticalcarrier should be any compatible, non-toxic Substance suitable todeliver the monoclonal antibodies to the patient. Sterile water,alcohol, fats, waxes, and inert solids may be used as the carrier.Pharmaceutically accepted adjuvants (buffering agents, dispersingagents) may also be incorporated into the pharmaceutical composition.Such compositions can contain a single monoclonal antibody so as to bespecific for two or more, but not all IATS serotypes of P. aeruginosa.Alternatively, a pharmaceutical composition can contain two or moremonoclonal antibodies to form a "cocktail." For example, a cocktailcontaining human monoclonal antibodies against groups of the various P.aeruginosa serotypes and immunotypes would be a universal product withactivity against the great majority of the clinical isolates of thatparticular bacterium.

Of interest are prophylactic and/or therapeutic monoclonal antibodycompositions capable of reacting with at least three IATS serotypes,usually at least four, and more usually at least five IATS serotypes. Ofparticular interest are monoclonal antibody compositions which reactwith at least about seven, preferably at least about ten to fourteen andup to and including all seventeen IATS serotypes. In conjunction withthe IATS serotypes, desirably the compositions will react with at leastone, usually at least two, and more usually at least three or four andup to and including all seven immunotypes of the Fisher immunotypingsystem.

Each of the compositions will include at least one, usually at leasttwo, and more usually at least three antibodies or more, where eachantibody reacts with at least 2, 3, 4, or more, but not all, IATSserotypes. Desirably there will be at least one monoclonal antibodywhich binds to at least two IATS serotypes and one or more Fisherimmunotypes, preferably at least two immunotypes.

Desirably, the total number of different monoclonal antibodies in acomposition will be at least one and equal to or less than aboutone-half the total number of IATS serotypes with which the compositionsreacts, frequently one-third such total number.

The mole ratio of the various monoclonal antibody components willusually not differ by more than a factor of 10, more usually by not morethan a factor of 5, and will usually be in a mole ratio of about 1:1-2to each of the other antibody components.

The human monoclonal antibodies of the present invention may also beused in combination with other monoclonal antibody compositions or withexisting blood plasma products, such as commercially available gammaglobulin and immune globulin products used in prophylactic ortherapeutic treatment of P. aeruginosa disease in humans. Preferably,for immune globulins the plasma will be obtained from human donorsexhibiting elevated levels of immunoglobulins reactive with P.aeruginosa. See generally, the compendium "Intravenous Immune Globulinand the Compromised Host," Amer. J. Med., 76(3a), Mar. 30, 1984, pgs1-231, which is incorporated herein by reference.

The monoclonal antibodies of the present invention can be used asseparately administered compositions given in conjunction withantibiotics or antimicrobial agents. Typically, the antimicrobial agentsmay include an anti-pseudomonal penicillin (e.g., carbenicillin) inconjunction with an aminoglycoside (e.g., gentamicin, tobramycin, etc.),but numerous additional agents (e.g., cephalosporins) well-known tothose skilled in the art may also be utilized.

The human monoclonal antibodies and pharmaceutical compositions thereofof this invention are particularly useful for oral or parenteraladministration. Preferably, the pharmaceutical compositions may beadministered parenterally, i.e., subcutaneously, intramuscularly orintravenously. Thus, this invention provides compositions for parenteraladministration which comprise a solution of the human monoclonalantibody or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.4% saline, 0.3% glycine and thelike. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, i.e.,from less than about 0.5%, usually at or at least about 1% to as much as15 or 20% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., preferably for the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intramuscular injectioncould be made up to contain 1 ml sterile buffered water, and 50 mg ofmonoclonal antibody. A typical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 150mg of monoclonal antibody. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pa.(1980), which is incorporated herein by reference.

The monoclonal antibodies of this invention can be lyophilized forstorage and reconstituted in a suitable carrier prior to use. Thistechnique has been shown to be effective with conventional immuneglobulins and art-known lyophilization and reconstitution techniques canbe employed. It will be appreciated by those skilled in the art thatlyophilization and reconstitution can lead to varying degrees ofantibody activity loss (e.g., with conventional immune globulins, IgMantibodies tend to have greater activity loss than IgG antibodies) andthat use levels may have to be adjusted to compensate.

The compositions containing the present human monoclonal antibodies or acocktail thereof can be administered for the prophylactic and/ortherapeutic treatment of P. aeruginosa infections. In therapeuticapplication, compositions are administered to a patient already infectedwith one or more P. aeruginosa serotypes, in an amount sufficient tocure or at least partially arrest the infection and its complications.An amount adequate to accomplish this is defined as a "therapeuticallyeffective dose." Amounts effective for this use will depend upon theseverity of the infection and the general state of the patient's ownimmune system, but generally range from about 1 to about 200 mg ofantibody per kilogram of body weight with dosages of from 5 to 25 mg perkilogram being more commonly used. It must be kept in mind that thematerials of this invention may generally be employed in serious diseasestates, that is life-threatening or potentially life-threateningsituations especially bacteremia and endotoxemia due to P. aeruginosa.In such cases, in view of the absence of extraneous substances and theabsence of "foreign substance" rejections which are achieved by thepresent human monoclonal antibodies of this invention, it is possibleand may be felt desirable by the treating physician to administersubstantial excesses of these antibodies.

In prophylactic applications, compositions containing the presentantibody or a cocktail thereof are administered to a patient not alreadyinfected by P. aeruginosa to enhance the patient's resistance to suchpotential infection. Such an amount is defined to be a "prophylacticallyeffective dose." In this use, the precise amounts again depend upon thepatient's state of health and general level of immunity, but generallyrange from 0.1 to 25 mg per kilogram, especially 0.5 to 2.5 mg perkilogram.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the antibody(ies) of this invention sufficient toeffectively treat the patient.

EXPERIMENTAL EXAMPLE I

Example I demonstrates methods for the production of human monocronalantibodies (IgM isotype) that react with IATS serotypes 2, 5, and 16 andFisher immunotypes 3 and 7.

A peripheral blood sample obtained from a cystic fibrosis patient knownto have had chronic infection with P. aeruginosa served as a source ofhuman B cells. Mononuclear cells were separated from the blood bystandard centrifugation techniques on Ficoll-Paque (Boyum, A., Scand. J.Clin. Lab. Invest. (1968) 21:Suppl. 97, 77-89) and washed twice incalcium/magnesium-free phosphate buffered saline (PBS).

The mononuclear cells were depleted of T-cells using a modifiedE-rosetting procedure. Briefly, the cells were first resuspended to aconcentration of 1×10⁷ cells/ml in PBS containing 20% fetal calf serum(FCS) at 4° C. One ml of this suspension was then placed in a 17×100 mmpolystyrene round bottom tube to which was added 1×10⁹2-amino-isothiouronium bromide (AET)-treated sheep red blood cells froma 10% (v/v) solution in Iscove's modified Dulbecco's medium (Iscove'smedium) (Madsen, M. and Johnson, H. E., J. Immun. Methods (1979)27:61-74). The suspension was very gently mixed for 5-10 minutes at 4°C. and the E-rosetted cells then removed by centrifugation onFicoll-Paque for 8 minutes at 2500×g at 4° C. E-rosette negativeperipheral blood mononuclear cells (E⁻ PBMC) banding at the interfacewere collected and washed once in Iscove's medium and resuspended insame containing 15% (v/v) FCS, L-glutamine (2 mmol/l) , penicillin (100IU/ml) , streptomycin (100 μg/ml) , hypoxanthine (1×10⁻⁴ M) ,aminopterin (4×10⁻⁷ M) , and thymidine (1.6×10⁻⁵ M). This medium ishereafter referred to as HAT medium.

Cell-driven transformation of the E⁻ PBMC was accomplished byco-cultivating these cells with a transforming cell line. Thetransforming cell line was an Epstein-Bart nuclear antigen (EBNA)positive human lymphoblastoid cell line derived by ethylmethanesulphonate (EMS) mutagenesis of the GM 1500 lymphoblastoid cellline followed by selection in the presence of 30μg/ml 6-thioguanine torender the cells hypoxanthineguanine phosphoribosyl transferase (HGPRT)deficient and thus HAT sensitive. This cell line is denominated the 1A2cell line and was deposited at the American Type Culture Collection(A.T.C.C.) on Mar. 29, 1982, under A.T.C.C. No. CRL 8119. 1A2 cells inlogarithmic growth phase were suspended in HAT medium and then combinedwith the E⁻ PBMC at a ratio of eight 1A2 cells per E⁻ PBMC. The cellmixture was plated into eight round-bottom 96-well microtiter plates(Costar 3799) at a concentration of 72,000 cells/well in a volume of 200μl per well, and incubated at 37° C. in a humidified atmospherecontaining 6% CO₂. Cultures were fed on days 5 and 8 post-plating byreplacement of half the supernatant with fresh HAT medium. The wellswere observed every other day on an inverted microscope for signs ofcell proliferation. Twelve days post plating, it was observed that 100%of the wells contained proliferating cells and that in most of thewells, the cells were of sufficient density for removal and testing ofsupernatants for anti-P. aeruginosa antibody.

Supernatants were screened for the presence of anti-P. aeruginosaantibodies using an enzyme linked immunosorbent assay (ELISA) technique(Engvall, E., (1977) 55:193-200). Antigen plates consisted offlat-bottom 96-well microtiter plates (Immulon II, Dynatech), the wellsof which contained various live bacteria adsorbed to the bottom of thewell. To facilitate adsorption of the bacteria to the plastic, 50μl/well of poly-L-lysine (PLL) (1 μg/ml in PBS, pH 7.2) was incubatedfor 30 minutes at room temperature. The unadsorbed PLL was then flickedout, the plates washed once with PBS, 50 μl of washed bacterialsuspension (OD₆₆₀ =0.2) in PBS added to each well, and the platesincubated for one hour at 37° C. Unadsorbed bacteria were removed bywashing the plates three times with saline-Tween [0.9% NaCl, 0.05% (v/v)Tween-20]. Various antigen plates used in the screen included: (1) amixture of P. aerucinosa Fisher immunotypes 1, 2, and 4 (A.T.C.C. Nos.27312, 27313, 27315); (2) a mixture of P. aeruginosa Fisher immunotypes3, 5, 6, and 7 (A.T.C.C. Nos. 27314, 27316, 27317, 27318, respectively);and (3) a microtiter plate with no bacteria.

The ELISA was initiated by first blocking the plates with 200 μl/well ofblocking buffer [PBS, pH 7.2, containing 5% (w/v) non-fat dry milk,0.01% (v/v) Antifoam A (Sigma), and 0.01% (w/v) thimerosal] for 60minutes at room temperature. After blocking, the plates were washedthree times with saline-Tween. Fifty μl of PBS, pH 7.2, containing 0.1%Tween-20 and 0.2% (w/v) bovine serum albumin (BSA) was then placed inall wells. Supernatants from wells of the culture plates were replicaplated into the corresponding wells of the antigen plates (50 μl/well)and the plates were incubated 30 minutes at room temperature.Supernatants were then removed, the wells washed three times withsaline-Tween, and 50 μl of appropriately diluted horseradish peroxidase(HRP) conjugated goat anti-human IgG+IgM (American Qualex International#A1114+#A1124) was added to the wells. In this example, HRP-goatanti-IgG and HRP-goat anti-IgM were used at a final dilution of 1:5000and 1:3000, respectively, in PBS, pH 7.2, containing 0.05% Tween-20 and0.1% BSA. Following a 30 minute incubation at room temperature, excessenzyme conjugated goat antibodies were removed and the wells were washedthree times with saline-Tween. One hundred μl of substrate (0.8 mg/mlo-phenylenediamine dihydrochloride in 100 mM citrate buffer, pH 5.0,plus 0.03% H₂ O₂ in deionized H₂ O, mixed in equal volumes just beforeplating) was then added to each well. After a 30 minute incubation inthe dark, 50 μl of 3N H₂ SO₄ was added to all wells to terminatereactions. Culture supernatants containing antibodies reacting with theplate's antigen were detected by positive color development in thecorresponding wells and the strength of the reaction quantitated bymeasuring the absorbency at 490 nm on a Bio-Tek EL-310 micro ELISAreader.

Analysis of the culture supernatants by the above method led to theidentification of two wells (1C1 and 2H12) which contained anti-P.aeruginosa antibodies that reacted with the Fisher immunotypes 3, 5, 6,and 7 plate, but not the Fisher immunotypes 1, 2, and 4 plate or theno-bacteria plate. In order to identify the specific Fisherimmunotype(s) recognized, antigen plates containing PLL-fixed bacteriaof only one Fisher immunotype were prepared as described above for eachimmunotype. Performance of the ELISA assay as set forth above withculture supernatants from wells 1C1 and 2H12 on the individualimmunotype plates indicated that these two wells contained antibodyspecific to both Fisher immunotype 3 and 7. A similar ELISA performed onthe IATS panel of P. aeruginosa typing strains (obtained from A.T.C.C.and included Nos. 33348-33364) demonstrated that antibody in wells 1C1and 2H12 reacted specifically with IATS strains 2, 5, and 16.

The isotype of the reactive antibody(s) in wells 1C1 and 2H12 wasdetermined in an ELISA assay similar to the specificity tests describedabove except that the HRP-goat anti-human IgG and HRP-goat anti-humanIgM were used independently as second step reagents, rather than beingcombined. Positive reaction of the antibody(s) in wells 1C1 and 2H12with Fisher immunotypes 3 and 7 was observed only with the anti-IgMreagents, demonstrating an IgM isotype for the relevant antibody(s) ineach well.

In order to determine if the anti-Fisher immunotypes 3 and 7 reactionpattern was due to one or more antibodies in wells 1C1 and 2H12 (i.e.,one antibody reactive with both Fisher immunotypes 3 and 7 or twoantibodies, one reactive with Fisher immunotype 3 and the other withFisher immunotype 7) cells from both wells were sub-cultured at lowdensity and wells containing proliferating cells assayed for antibodyactivity on separate Fisher immunotype 3 and Fisher immunotype 7bacteria antigan plates. Subculture was performed in 96-well roundbottom plates at a density of 5 cells/well in a total volume of 100 μlof HAT medium lacking the aminopterin component (HT-medium).Non-transforming, HAT-sensitive lymphoblastoid cells were included inall wells at a density of 500 cells/well as feeder cells. Four dayspost-plating, 100 μl of HAT-medium was added to all wells to selectivelykill the feeder cells. Wells were again fed on day 9 postplating byreplacement of half the supernatant with HAT medium. Thereafter, cellswere similarly fed every 4-5 days with HT-medium until wells were ofsufficient lymphoblastoid cell density for supernatant analysis byELISA. When assayed on individual Fisher immunotype 3 and Fisherimmunotype 7 antigen plates, all those supernatants that reacted withFisher immunotype 3 also reacted with Fisher immunotype 7, indicatingthat one antibody was responsible for the activity on both Fisherimmunotypes. Randomly selected supernatants when assayed on IATS strains2, 5, and 16 were found to react with all three strains rather thanindividual strains, further supporting the conclusion that one antibodywas cross-reacting with Fisher immunotypes 3 and 7 and IATS strains 2,5, and 16.

Cloning of specific antibody-producing cells from wells 1C1 and 2H12 wasaccomplished by independently subjecting the cells from each well toseveral rounds of limiting dilution cloning until all clonalsupernatants assayed by the above ELISA protocol resulted in a positivereaction on Fisher immunotypes 3 and 7 and IATS strains 2, 5, and 16.Cloning employed feeder cells as described above for subculturing. Bythese means, two cloned transformed human cell lines (1C1 and 2H12) wereachieved which are continuous (immortal) and which each secrete humanmonoclonal antibody reactive with Fisher immunotypes 3 and 7 and IATSstrains 2, 5, and 16. In this example, the cell line and antibody itproduces carry the same designation.

EXAMPLE II

Example II demonstrates methods for the production of human monoclonalantibody (IgG isotype) that reacts with IATS serotypes 2, 5 and 16 andFisher immunotypes 3 and 7. The protocols for the production areessentially identical to Example I. Briefly, a peripheral blood sample,obtained from a cystic fibrosis patient known to have had chronicinfection with P. aeruginosa, served as a source for human B cells.Mononuclear cells were separated as described in Example I, except that1.6 mls of the PBS suspension were used with 1.6×10⁹ AET-treated sheepred blood cell suspension. Cell driven transformation was also the same,except that: the ratio of 1A2 cells per E⁻ PBMC was 7.5; fifteen platesat 17,000 cells/well were used; cultures were fed at6 and 10 dayspost-plating; and at sixteen days post-plating, nearly all of the wellscontained proliferating cells.

Screening of the culture supernatants for specific antibodies wasperformed as described, and resulted in locating one well (9D1) thatcontained an antibody reactive with the Fisher immunotypes 3, 5, 6, and7 plate, but not the Fisher immunotypes 1, 2 and 4 plate or theno-bacteria plate. Performance of the described ELISA assay onindividual immunotype or serotype bacterial antigen plates with 9D1culture supernatants indicated an antibody specific to both Fisherimmunotypes 3 and 7, as well as IATS strains 2, 5 and 16. Subsequentstudies performed in accordance with Example I demonstrated that theantibody specificity in well 9D1 was attributable to a single clonesecreting IgG isotype antibody.

EXAMPLE III

Example III demonstrates the antigenic specificity of some of theantibodies of the present invention.

To determine if monoclonal antibodies 1C1, 2H12, and 9D1 reacted withthe same antigenic target and were thus identical in specificity,additional assays were performed. First, the antibodies were compared inan ELISA on a panel of reference strains and clinical isolates of P.aeruginosa. The ELISA protocol was as described above except for thefollowing modifications: 1) Instead of bacteria adsorbed to PLL coatedplates, the wells of the plate contained various whole bacteria that hadbeen ethanol-fixed to the bottom of the well. Plates were prepared byaddition of 50 μl of washed bacterial suspension (OD₆₆₀ =0.2) in PBSinto the wells, centrifugation of the plates for 20 minutes at 500×g,aspiration of PBS, addition of 75 μl of ethanol for 10 minutes, removalof ethanol, followed by air drying. Included on the antigen plates wereIATS strains 2, 5, 11, and 16 (A.T.C.C. Nos. 33349, 33352, 33358, and33363 respectively) and sixteen clinical isolates that had previouslybeen typed by both agglutination with Difco Detroit, Mich.]Bacto-Pseudomonas aeruginosa antisera according to the manufacturer'sdirections and by ELISA (as described herein) as serotypes 2, 5, 16, ora combination of such serotypes; 2) Rabbit typing antisera were used ata dilution of 1:500 in PBS except for anti-IATS 16 which was diluted1:250. Culture supernatants containing 1C1, 2H12 and 9D1 antibodies wereused neat; 3) As second step reagents, biotinylated protein A (B-2001,Vector Laboratories, Inc., Burlingame, Calif.) diluted 1:500 andbiotinylated goat anti-human Ig (4703, Tago, Inc., Burlingame, Calif.)diluted 1:500, were used for detection of rabbit and human antibodiesrespectively. Fifty μl of reagent were added to appropriate wells andafter a 30 minute incubation at room temperature, unbound reagent wasremoved and the wells were washed three times. Fifty μl of a preformedavidin:biotinylated horseradish peroxidase complex (Vectastain ABC Kit,Vector Laboratories, Inc., Burlingame, Calif.) was then added to eachwell. After a 30 minute incubation at room temperature, the excessVectastain ABC reagent was removed, and the wells again washed threetimes before the addition of substrate.

As shown in Table II, results of the assay indicated that whereasantibodies 1C1 and 9D1 reacted with every clinical isolate typed as anIATS 2, 5, or 16 serotype, antibody 2H12 failed to react with three suchisolates. This suggests that antibody 2H12 recognized a differentepitope from that recognized by 1C1 or 9D1 and that the two epitopes areapparently coordinately expressed on most but not all clinical isolatescorresponding to IATS serotypes 2, 5, or 16.

                  TABLE II                                                        ______________________________________                                        REACTIVITY PATTERNS OF DIFCO BACTO-P.                                         AERUGINOSA ANTISERA AND HUMAN MONOCLONAL                                      ANTIBODIES 2H12, 1C1, AND 9D1 ON TYPE STRAINS                                 AND CLINICAL ISOLATES OF P. AERUGINOSA                                        Type                                                                          Strain  Rabbit              Human                                             or Clinical                                                                           IATS    IATS    IATS  IATS  MAb  MAb  MAb                             Isolate α2                                                                              α5                                                                              α16                                                                           α11                                                                           2H12 1C1  9D1                             ______________________________________                                        IATS 2  +       -       -     -     +    +    +                               IATS 5  (+).sup.a                                                                             +       -     -     +    +    +                               IATS 16 (+).sup.a                                                                             (+).sup.a                                                                             +     -     +    +    +                               IATS 11 -       -       -     +     -    -    -                               A523    +       +       -     -     +    +    +                               B406    +       +       (+).sup.a                                                                           -     +    +    +                               C27     +       +       -     -     +    +    +                               D26     +       +       -     -     +    +    +                               F155    +       +       +     -     +    +    +                               F225    +       (+).sup.a                                                                             +     -     +    +    +                               F250    +       +       -     -     +    +    +                               F253    -       +       -     -     +    +    +                               F255    +       +       -     -     -    +    +                               F256    +       -       -     -     -    +    +                               F396    +       +       +     -     +    +    +                               H217    +       +       +     -     +    +    +                               H218    +       +       -     -     +    +    +                               H219    -       +       -     -     +    +    +                               H220    +       +       -     -     +    +    +                               H221    +       +       -     -     -    +    +                               ______________________________________                                         .sup.a (+) =  ELISA reaction very weakly positive                        

The data further suggested that the molecular target recognized byantibodies 1C1 and 9D1 was likely to be present on all clinical isolatesof P. aeruginosa that could be typed as belonging to IATS serotypes 2,5, or 16 while the target recognized by antibody 2H12 was expressed on asubgroup of such isolates.

To determine whether the 1C1, 2H12, and 9D1 antibodies reacted withdifferent antigens or alternatively, different epitopes on the sameantigen with such epitopes expressed on most but not all IATS 2, 5, and16 serotypes, immunoblot analysis was performed. Because the sharedantigenicity between IATS strains 2, 5, and 16 is apparently due to theheat stable antigens (Liu, P. V. et al., supra) and in keeping with thefact that heat-stability is a previously noted characteristic oflipopolysaccharide molecules, LPS preparations from IATS strains 2, 5,16 and 11 were chosen as antigen preparations for analysis. Crude LPSwas prepared from the type strains by extraction in saline at 60° C.(Orskov, F. et al., Acta. Path. Microbiol. Scand. (1971) Section B79:142-152). Ten micrograms of LPS, as determined by2-keto-3-deoxyoctonate (KDO) content (Karkhanis, Y. D., et al., Anal.Biochem. (1978) 85:595-601) from each of the serotypes were subjected tosodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)(Hancock, R. E. W. and Carey, A. M., J. Bacteriol. (1977) 140:901-910)on a 10-20% gradient gel. Separated molecular species were transferredfrom the gel to a nitrocellulose membrane (NCM) as described elsewhere(Towbin, H., et al., Proc. Natl. Acad. Sci. USA (1979) 76:4350-4354) andthe NCM blot blocked for 1 hr in PBS-Tween (Batteiger, B., et al., J.Immunol. Meth. (1982) 55:297-307). The blots were then incubated for 1hr at room temperature in 20 ml of spent culture supernatant from eitherthe 1C1, 2H12, or 9D1 cell lines. Following five 5 minute rinses inPBS-Tween, each of the NCM blots was incubated for 1 hr at 25° C. in a1:1000 or 1:1500 dilution (in PBS-Tween) of alkalinephosphatase-conjugated goat anti-human IgG+IgA+IgM (Zymed). The blotswere then subjected to five 5-minute washes in PBS-Tween after whichtime antigen-antibody interactions were visualized by incubating theblots for 15-20 minutes at 25° C. in 30 ml ofnitroblue-tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP)substrate as described by Leary et al., Proc. Natl. Acad. Sci. USA(1983) 80:4045-4049. Color development was stopped by rinsing the blotseveral times in deionized water.

The blot profiles obtained with the three antibodies were notablydifferent. Antibody 2H12 predominantly recognized a short series ofregularly spaced (i.e., ladder-like) low molecular weight molecules inthe antigen preparations of serotypes 2, 5, and 16. Some regularlyspaced higher molecular weight molecules were also faintly recognized.No reaction was observed on the LPS preparation from serotype 11. Arepeat of the immunoblot analysis using LPS preparations that hadpreviously been treated with proteinase K at 60° C. to destroy proteinantigens resulted in no alterations in profiles. The low molecularweight bands corresponded precisely with smaller forms of LPS moleculesas visualized in a similarly performed SDS-PAGE gel in which theantigens were not transferred to a NCM but were instead specificallystained for the presence of LPS (Tsai, C. M. and Frasch, C. E., Anal.Biochem. (1982) 119:115-119) except that the lowest band on the silverstained gel (representing the core region plus lipid A of LPS) was notrecognized. Antibody 1Cl recognized the same series of regularly spacedlow molecular weight molecules among antigen preparations of serotypes2, 5, and 16 but not 11, although the intensity of reaction was not asstrong as that with 2H12. In addition, however, this antibody alsoprominently recognized a series of regularly spaced higher molecularweight molecules on serotypes 2, 5, and 16 which in combination with therecognized lower molecular weight bands, gave rise to distinct, fullladder-like profiles. These profiles were not altered by pretreatment ofthe antigen preparations with proteinase K. Again, the profilescorresponded to the ladder-like banding patterns observed inLPS-specific stained gels (i.e., they appeared to correspond band forband) except that the band representing core plus lipid A was notrecognized. Antibody 9D1 had the same reaction profile as antibody 1Clon the IATS 2, 5, and 16, but not 11, strains. Again, the bottom-mostband of the ladder of a silver stained gel of the different LPSpreparations was not recognized and the overall profile was not alteredby pretreatment of the LPS preparations with proteinase K. Collectively,these observations indicated that the LPS of serotypes 2, 5, and 16 wasthe molecular target recognized by the 2H12, 1C1, and 9D1 antibodies.

EXAMPLE IV

Example IV demonstrates the protective activity of one of the antibodiesof the present invention.

To assess the in vivo protective capacity of antibody 1C1, animalprotection studies were performed in mice. 1C1 antibody was firstconcentrated from spent culture supernatant by precipitation withsaturated ammonium sulphate (50% final concentration) (Good, A. H., etal., Selected Methods in Cellular Immunology, Mishell, B. B. and Shiigi,S. M., eds., W. J. Freeman & Co., San Francisco, Calif., 279-286(1980)). Precipitated material was reconstituted in a minimum volume ofsterile water, extensively dialyzed against PBS, and sterile filtered.As a negative control, spent culture supernatant from anothertransformed human cell line (6F11-A.T.C.C. No. CRL 8562) producing ahuman monoclonal antibody specific for the LPS of IATS strain 11 wassimilarly treated.

Female BALB/c mice between 20 and 22 grams body weight were divided intotwo groups of thirty mice each. All mice in each group were individuallyinoculated by the intraperitoneal (ip) route with 0.5 ml of concentrated1Cl or 6F11 antibody. Six hours later each of the two groups wassubdivided into three groups of ten mice and members of each ten micegroup were independently challenged ip with 0.3 ml of a live bacterialsuspension containing 5 LD50 of IATS strain 2, 5, or 11, respectively.Bacterial suspensions were prepared from a broth culture in logarithmicphase growth, from which the bacteria were centrifuged, washed twice inPBS and resuspended to the appropriate density in PBS. Control groupsconsisting of four or five mice each were injected intraperitoneallywith 0.5 ml of PBS and six hours later challenged intraperitoneally with5 LD₅₀ of the same serotypes. After bacterial challenge, animals werethen observed for a period of five days.

As shown in Table III, antibody 1C1 provided specific and significantprotection against a lethal challenge of both IATS 2 and IATS 5serotypes, but not the IATS 11 serotype. The typical course followingbacterial challenge in all unprotected animals involved varying periodsof endotoxic shock and was usually followed by death. Control animalsgiven only PBS went through acute endotoxic shock and all proceededrapidly to death. Negative control animals given non-homologous antibody(6F11) had a period of more prolonged shock characterized by thesymptoms listed in footnote "a" to Table III. Some of these animalseventually recovered, possibly due to the non-antibody components thatwere co-concentrated in the antibody preparations. The animals whichreceived the protective homologous antibody, however, displayed onlyminor symptoms of endotoxemia. These symptoms disappeared within 24hours of innoculation, and the animals then appeared healthy throughoutthe remainder of the five day observation period.

                  TABLE III                                                       ______________________________________                                        In Vivo Demonstration of the Protective Effect of                             Human Monoclonal Antibody 1C1 Against                                         IATS Serotypes 2 and 5                                                        Human     No. Survivors/No. of Animals Challenged                             Monoclonal                                                                              Five Days After Challenge With:                                     Antibody  IATS 2      IATS 5     IATS 11                                      ______________________________________                                        1C1       8/10        10/10      3/10.sup.a                                   6F11      0/10        1/10.sup.a 10/10                                        PBS       0/4         0/4        0/4                                          ______________________________________                                         .sup.a In those cases where nonspecific protection was noted, recovery        from infection was markedly different from that seen between homologous       antibodies and infecting strains, i.e., 1C1 with IATS 2 or 5 and 6F11 wit     IATS 11. In the latter cases, recovery was essentially complete with          animals appearing normal 24 hours after the bacterial challenge. In           nonspecific cases, however, mice exhibited signs of acute infection (i.e.     diarrhea, crusted eyelids, ruffled fur, "hunched" profile, and slow           movement) for several days before recovering to normal status. Such           nonspecific protection is apparently due to nonantibody components that       are coconcentrated and thus injected into the animals, since all animals      given only PBS died.                                                     

Utilizing the same protocol, a second experiment was performed in whichgroups of mice were challenged with Fisher strains 2, 3 or 7 and theanimals observed for five days. As shown in Table IV, antibody 1C1 againprovided specific and significant protection against an otherwise lethalinfection with Fisher immunotypes 3 and 7, but was ineffective againstFisher immunotype 2. The cell line 1C1 was deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, onNov. 8, 1985 and assigned accession number ATCC No. CRL 8941.

                  TABLE IV                                                        ______________________________________                                        In Vivo Demonstration of the Protective Effect                                of Human Monoclonal Antibody lCl Against                                      Fisher Immunotypes 3 and 7                                                    Human     No. Survivors/No. of Animals Challenged                             Monoclonal                                                                              Five Days After Challenge With:                                     Antibody  Fisher 3    Fisher 7   Fisher 2                                     ______________________________________                                        1C1       10/10       10/10      1/10.sup.a                                   6F11      0/10        7/10.sup.a 10/10                                        PBS       0/5         0/5        0/5                                          ______________________________________                                         .sup.a Survival most likely due to nonspecific protection as described in     the footnote to Table III.                                               

EXAMPLE V

Example V demonstrates a method for the production of a human monoclonalantibody that reacts with IATS serotypes 4 and 11 and Fisher immunotype2 of P. aeruginosa. The process described in Examples I-IV was repeatedexcept that it was necessary to make certain modifications to isolate,characterize and assay the antibody described in this Example. Thefollowing are the changes in the procedure and the results obtained withthe monoclonal antibody described herein.

The source of human B cells was an individual previously immunized witha high molecular weight polysaccharide preparation isolated from Fisherimmunotype (Pier, G. B., et al., Infec. Immun. [1984] 45:309-313, whichis incorporated herein by reference).

High-molecular-weight polysaccharide was prepared as previouslydescribed (Pier, J. Clin. Invest. 69:303-308 (1982), Pier et al.,Infect. Immun. 42:936-941 (1983), and Pier et al., Infect. Immun.34:461-468 (1981). The organisms were grown in 14 liters of thechemically defined medium of Terleckyj et al., Infect. Immun. 11:64914655 (1975), in an LSL Biolafitte 20-liter fermentor. The LPS wasextracted from lyophilized cells of this IT-3 strain by the phenol-watermethod of Westphal et al., Z. Naturforsch. 79:148-155 (1952) andpurified as previously described (Pier et al., 1981, supra, and Pier etal., Infect. Immun, 22:908-918 (1978). O polysaccharide side chains werederived from the LPS by hydrolysis in 1% acetic acid at 95° C. for 6 h.The lipid A precipitate was removed by centrifugation, and thesupernatant was dialyzed against deionized water and lyophilized.

Cell-driven transformation of the E⁻ PBMC was accomplished byco-cultivating these cells with the transforming cell line 1A2. 1A2cells in logarithmic growth phase were suspended in HAT medium and thencombined with the E⁻ PBMC at a ratio of fifteen 1A2 cells per E⁻ PBMC.The cell mixture was plated into fourteen microtiter plates at aconcentration of 62,000 cells/well in a volume of 200 μl per well.Cultures were fed on days 7 and 11 post-plating, and by day 15 it wasobserved that 100% of the wells contained proliferating cells.

To screen for the presence of anti-P. aeruginosa antibodies, two antigenplates were used which consisted of: (1) a mixture of P. aeruginosaFisher immunotypes 1 through 7 (A.T.C.C. Nos. 27312, 27313, 27314,27315, 27316, ⁻²⁷³¹⁷ and 27318, respectively); and (2) a PLL-treatedmicrotiter plate with no bacteria.

Analysis of the culture supernatants by the method of the Examples aboveled to the identification of approximately 100 wells which containedanti-P. aeruginosa antibodies reactive with the Fisher immunotypes 1-7plate, but not the PLL-treated plate that lacked bacteria. In order toidentify the specific Fisher inununotype(s) recognized, antigen plateswere constructed as above in which each row of the plates containedPLL-fixed bacteria of only one Fisher immunotype. An ELISA was performedas set forth above, with culture supernatant from each of the anti-P.aeruginosa positive wells placed in a columnar array on the new antigenplates resulting in the identification of a number of wells thatcontained antibody specific for Fisher immunotype 2. To further analyzethe serological specificity of the anti-Fisher immunotype 2 antibodies,the subernatants were tested in a similar ELISA on antigen platesconstructed to contain each of the seventeen IATS serotypes of P.aeruginosa in separate wells. Results of this assay indicated that thegreat majority of the supernatants were specific for IATS serotype 11although one supernatant, in well 6D6, demonstrated a cross-reactivespecificity pattern on IATS serotypes 4, 11, 13, and 14.

In order to determine if the anti-IATS serotypes 4, 11, 13, and 14reaction pattern was due to one or multiple antibodies in well 6D6,additional aliquots of supernatant from well 6D6 were independentlyadsorbed with IATS serotypes 4, 7 (negative control), 11, 13, and 14 andthe adsorbed supernatants then tested on PLL-fixed bacteria of each ofthe five IATS serotypes according to the ELISA assay outlined above.Adsorptions were performed by resuspending a packed bacterial cellpellet with an equal volume of supernatant for one half hour on icefollowed by separation of the supernatant from the bacteria bycentrifugation. Results of this assay indicated that IATS serotypes 4and 11 adsorbed out antibody activity against each other, but notagainst IATS serotypes 7, 13, or 14. This demonstrated the presence ofat least two different anti-P. aeruginosa antibodies in well 6D6, atleast one of which cross-reacted with IATS serotypes 4 and 11.

Isolation and cloning of the appropriate antibody-producing cells fromwell 6D6 was accomplished in three steps. The first step involved lowdensity subculture of the cells at 20 cells/well and was followed byanother round of low density subculture (5 cells/well) of cells obtainedfrom an anti-IATS serotypes 4+11 positive well which had been generatedin the first 20 cells/well round of low density subculture. Each roundof subculture was performed in 96-well round bottom plates at the stateddensity in a total volume of 100 μl of HAT medium lacking theaminopterin component (HT-medium). Non-transforming, HAT-sensitivelymphoblastoid cells were included in all wells at a density of 500cells/well as feeder cells. Four days post-plating, 100 μl of HAT-mediumwas added to all wells to selectively kill the feeder cells. Wells wereagain fed on day 9 post-plating by replacement of half the supernatantwith HAT medium. Thereafter, wells were similarly fed every 4-5 dayswith HT-medium until wells were of sufficient lymphoblastoid celldensity for supernatant analysis by ELISA as described earlier.

In each assay, those supernatants that were reactive with IATS serotype4 were also reactive with IATS serotype 11 and Fisher immunotype 2. Inaddition, antibody activity against IATS serotypes 13 and 14 was lost,thus confirming the earlier specificity assignments.

Formal cloning of specific antibody-producing cells was performed byfirst plating the cells at very low density (calculated 1/well) into72-well Terasaki plates (Nunc #1-36538) in a volume of 10 μl/well.Plates were placed in an incubator for 3 hours to allow the cells tosettle to the bottom of the plate and then microscopically scored by twodifferent individuals for wells containing a single cell. Each of thesecells was then independently placed into the wells of a 96-well roundbottom plate with feeder cells and cultured as outlined earlier for lowdensity subculture. Supernatants from all arising clones when assayed bythe above ELISA protocol were positive for anti-IATS serotypes 4 and 11.

By these means a cloned transformed human cell line was achieved whichgrows continuously (is immortal) and secretes human monoclonal antibodyreactive with Fisher immunotype 2 and cross-reactive with IATS serotypes4 and 11. In this example, the cell line and antibody it produces carrythe same designation (i.e., 6D6).

The isotype of the antibody in well 6D6 was determined in an ELISA assaysimilar to the specificity tests described above except that HRP-goatanti-human IgG and HRP-goat anti-human IgM were used independently assecond step reagents, rather than being combined. Positive reaction ofthe antibody in well 6D6 with Fisher immunotype 2 and IATS serotypes 4and 11 was observed only with the anti-IgM reagent, demonstrating an IgMisotype for this antibody.

Biochemical characterization of the molecular species recognized by the6D6 antibody was accomplished by immunoblot analysis as described inExample 3 above, except that LPS preparations from Fisher immunotype 2and IATS serotypes 4 and 11 were chosen as antigen preparations foranalysis. An LPS preparation from Fisher immunotype 1 was included as anegative control.

Prior to analysis, each of the crude LPS preparations was diluted 1:1 indissociation buffer [0.125M Tris, 4% (w/v) sodium dodecyl sulphate(SDS), 20% (v/v) glycerol, 10% (v/v) β-mercaptoethanol, 0.4% (w/v)bromphenol blue, pH 6.8] and bath sonicated for 5 minutes. In order todegrade proteins in the samples, proteinase K (1 mg/ml in H₂ O) wasadded to each sample in a 40% (w/w) ratio of enzyme to LPS and incubatedat 60° C. for 2 hours with a 5-minute bath sonication step after 1 hour.The samples were then heated to 100° C. for 5 minutes and centrifuged ina microfuge for 2 minutes.

Clarified samples representing 10 μg of LPS from each of the bacterialstrains were subjected to SDS polyacrylamide gel electrophoresis(SDS-PAGE) as described above. After transferring the separated moleculespecies to a NCM, the NCMs were then incubated for 2 hours at roomtemperature in 10 ml of spent culture supernatant from the 6D6 cellline. The remainder of the procedure was as described above.

Positive results were noted only in the tracks that contained Fisherimmunotype 2, IATS serotype 4, or IATS serotype 11 LPS. In the Fisherimmunotype 2 and IATS serotype 11 tracks, antibody 6D6 recognized ashort series of regularly spaced (i.e., ladder-like) low molecularweight molecules which corresponded precisely with smaller forms of LPSmolecules as visualized in a similarly performed SDS-PAGE gel in whichthe antigens were not transferred to a NCM, but were insteadspecifically stained for the presence of LPS, except that the lowestband on the silver stained gel (representing the core region plus lipidA of LPS) was not recognized. In contrast, in the lane containing IATSserotype 4 LPS, antibody 6D6 recognized a full series of regularlyspaced bands nearly spanning the full length of the gel, with the mostintense reaction occurring among the higher molecular weight bands.Again, this profile corresponded to the ladder-like banding patternobserved in the LPS-specific stained gel (i.e., they appeared tocorrespond band for band), with the exception that the band representingcore plus lipid A was not recognized. These data clearly indicated thatthe O-side chain of LPS was the molecular target recognized by antibody6D6 on Fisher immunotype 2 and IATS serotypes 4 and 11.

To assess the in vivo protective capacity of antibody 6D6, animalprotection studies were performed in mice. The 6D6 antibody was firstconcentrated from spent culture supernatant by precipitation withsaturated ammonium sulphate (50% final concentration). Precipitatedmaterial was reconstituted in a minimum volume of sterile water,extensively dialyzed against PBS, and sterile filtered. As a negativecontrol, spent culture supernatant from another transformed human cellline (C5B7-A.T.C.C. No. CRL 8753) producing a human monoclonal antibodyspecific for the LPS of Fisher immunotype 1 was treated in the samemanner. Similarly, as a positive control, spent culture supernatant fromtransformed human cell line 6F11 (A.T.C.C. No. CRL 8562) producing ahuman monoclonal antibody specific for the LPS of Fisher immunotype 2and IATS serotype 11 was concentrated.

Female Swiss-Webster mice between 20 and 22 grams body weight weredivided into three groups of twenty mice each. All mice in each groupwere individually inoculated by the intraperitoneal (ip) route with 0.5ml of concentrated 6D6, C5B7, or 6F11 antibody. Six hours later, each ofthe three groups was subdivided into four groups of five mice andmembers of each five-mice group were independently challenged ip with0.3 ml of a live bacterial suspension containing 8 LD₅₀ of Fisherimmunotype 1, Fisher immunotype 2, IATS serotype 4 or IATS serotype 11,respectively. Bacterial suspensions were prepared as described above inExample IV. After bacterial challenge, the animals were observed for aperiod of five days. Results were as follows:

                  TABLE V                                                         ______________________________________                                        In Vivo Demonstration of the Protective Effect of                             Human Monoclonal Antibody 6D6 Against                                         Fisher Immunotype 2 and IATS Serotypes 4 and 11                               Human     No. Survivors/No. Tested                                            Monoclonal                                                                              Five Days After Challenge With:                                     Antibody  Fisher 1  Fisher 2  IATS 4 IATS 11                                  ______________________________________                                        6D6       0/5       5/5       4/5    5/5                                      6F11      0/5       5/5       0/5    5/5                                      C5B7      5/5       1/5       0/5    0/5                                      ______________________________________                                    

As shown in Table V, antibody 6D6 provided specific and significantprotection against a lethal challenge of Fisher immunotype 2, IATSserotype 4 , and IATS serotype 11, but not Fisher immunotype 1. The cellline 6D6 was deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, on Aug. 8, 1986 and assignedaccession number ATCC No. CRL 9171.

EXAMPLE VI

Example VI demonstrates a method for the production of a humanmonoclonal antibody that reacts with IATS serotypes 6 and 13 and Fisherimmunotype 1 of P. aeruginosa. The process described in Examples Ithrough V was repeated, except that it was necessary to make certainmodifications to isolate, characterize and assay the antibody describedin this Example. The following are the changes in the procedures and theresults obtained with the monoclonal antibody described herein.

The source of human B cells was an individual previously immunized witha high molecular weight polysaccharide preparation isolated from Fisherimmunotype 2 (Pier et al., Infec. Immun., 34:461 (1981)). Aftercollecting the E⁻ PBMC, as described above, the cells were frozen in FCScontaining 10% (v/v) DMSO in a liquid nitrogen vapor tank. These cellswere later thawed quickly at 37° C., washed once in Iscove's medium andresuspended in HAT medium. Cell-driven transformation was accomplishedat a ratio of 15 1A2 to cells per E⁻ PBMC. The cell mixture plated into20 microtiter plates at a concentration of 78,500 cells/well. Cultureswere fed every 3-5 days post-plating and at eleven days it was observedthat 100% of the wells contained proliferating cells.

To screen supernatants for the presence of anti-P. aeruginosa antibodiesusing the ELISA technique, the antigen plate consisted of a mixture ofP. aeruginosa Fisher immunotypes 1-7 [clinical isolate PSA I277 (GeneticSystems Corporation Organism Bank ("GSCOB")), ATCC 27313, PSA G98(GSCOB), ATCC 27315, PSA F625 (GSCOB), ATCC 27317 and ATCC 27318,respectively]. A PLL-treated microtiter plate with no bacteria was alsoused in the screen.

Analysis of the culture supernatants by the above method lead to theidentification of approximately 200 wells which contained anti-P.aeruginosa antibodies reactive with the Fisher immunotypes 1-7 plate,but not the PLL-treated plate that lacked bacteria. In order to identifythose wells that contained antibody reactive with two or more IATSserotypes, antigen plates were constructed, as above, in which a columnof the plates contained PLL-fixed bacteria of only one IATS serotype. AnELISA was performed, as set forth above, with supernatant from anexpanded culture of each of the anti-P. aeruginosa positive wells.Supernatants were placed in a row on the new antigen plates and resultedin the identification of a number of wells that contained antibodyreactive with multiple IATS serotypes. One well, designated 8H7,contained antibodies specific for IATS serotypes 6 and 13. When thesupernatant from this well was tested by ELISA on the 7 Fisherimmunotypes, as in Example V, it was demonstrated that the antibodies of8H7 were specific for Fisher immunotype 1.

In order to determine if the anti-IATS serotypes 6 and 13 reactionpattern was due to one of multiple antibodies in well 8H7, additionalaliquots of supernatant were independently adsorbed with IATS serotypes6, 13, and 17 (negative control), and the adsorbed supernatants thentested on PLL-fixed bacteria of each of the three IATS serotypesaccording to the ELISA assay outlined above. Results indicated that IATSserotypes 6 and 13 adsorbed out antibody activity against each other.IATS serotype 17 adsorbed out no activity against either IATS 6 or 13.This data demonstrated that the anti-IATS serotype 6 and 13 reactionpattern was due to a single antibody from well 8H7.

Isolation and cloning of the antibody-producing cells from well 8H7 wasaccomplished in three steps, essentially as described in Example Vabove. By these means a cloned transformed human cell line was achievedwhich grows continuously (i.e., is immortal) and secretes humanmonoclonal antibody reactive with Fisher immunotype 1 and cross-reactivewith IATS serotypes 6 and 13. In this example, the cell line andantibody it produces carry the same designation, 8H7. Using a proceduresimilar to that described in Example V, the isotype of the antibody inwell 8H7 was determined to be IgM.

Biochemical characterization of the molecular species recognized by the8H7 antibody was accomplished by immunoblot analysis as described inExamples III and V above, except that the LPS preparations from Fisherimmunotype 1 and IATS serotypes 6 and 13 were chosen as antigenpreparations for analysis. An LPS preparation from IATS serotype 10 wasincluded as a negative control.

Analysis of the resulting immunoblots showed positive results only inthe NCM tracks that contained Fisher immunotype 1, IATS serotype 6, orIATS serotype 13 LPS. In the Fisher immunotype 1 and IATS serotype 6tracks, antibody 8H7 recognized a series of regularly spaced (i.e.,ladder-like) bands spanning nearly the full length of the gel. Thesebands corresponded precisely with the multiple molecular weight forms ofLPS molecules, as visualized in a similarly performed SDS-PAGE gel inwhich the antigens were not transferred to a NCM, but were insteadspecifically stained for the presence of LPS. In the lane containingIATS serotype 13 LPS, antibody 8H7 recognized a more abbreviated seriesof regularly spaced bands confined to the mid-to-upper molecular weightrange of the gel. Again, the bands which were recognized corresponded inposition to those observed in an LPS-specific stained gel, except thatthe highest and lowest molecular weight forms of LPS in the stained geldid not appear to be well-recognized in the Western blot. These dataclearly indicated that LPS was the molecular target recognized byantibody 8H7 on Fisher immunotype 1 and IATS serotypes 6 and 13.

To assess the in vivo protective capacity of antibody 8H7, animalprotection studies were performed in mice, as described in Examples IVand V above. As a negative control, spent culture supernatant fromanother transformed human cell line (6F11-ATCC No. CRL 8652), producinga human monoclonal antibody specific for the LPS of Fisher immunotype 2,was treated in the same manner. As a positive control, spent culturesupernatant from transformed human cell line C5B7 (ATCC No. CRL 8753),producing a human monoclonal antibody specific for the LPS of Fisherimmunotype 1 and IATS serotype 6, was used.

Female Swiss-Webster mice between 20 and 22 grams body weight weredivided into three groups of 30 mice each. All mice in each group wereindividually inoculated intraperitoneally (ip) with 0.5 ml ofconcentrated 8H7, 6F11 or C5B7 antibody. Four hours later, each of thethree groups was subdivided into three groups of ten mice and members ofeach ten-mice group were independently challenged ip with 0.3 ml of alive bacterial suspension containing 9.4LD₅₀ of a clinical isolate(A522) representative of the IATS 6 serotype (Fisher immunotype 1equivalent), 5LD₅₀ of the IATS 13 reference serotype, or 10LD₅₀ of theIATS 11 (Fisher inununotype 2 equivalent) reference serotype. Bacterialsuspensions were prepared as described above in Example IV. Afterbacterial challenge, the animals were observed for a period of fivedays. Results were as follows:

                  TABLE VI                                                        ______________________________________                                        In Vivo Demonstration of the Protective Effect of Human                       Monoclonal Antibody 8H7 Against IATS Serotypes 6 and 13                       Human      Number Survivors/Number Tested,                                    Monoclonal Five Days After Challenge With:                                    Antibody   IATS 6      IATS 13  IATS 11                                       ______________________________________                                        8H7        9/10        6/10     1/10                                          C5B7       9/10        0/10     0/10                                          6F11       0/10        2/10     10/10                                         ______________________________________                                    

As shown in Table VI, antibody 8H7 provided specific and significantprotection against a lethal challenge of IATS serotype 6, but not IATSserotype 11. The protection against IATS serotype 13 was to a lesserdegree, but may be explained by the relatively high number of organismsrequired to achieve the LD₅₀ for this isolate when compared to theserotype 6 isolate (3×10⁷ colony forming units and 2.6×10⁶ colonyforming units, respectively). In a separate experiment, antibody; 8H7protected five of five mice challenged with 3.5LD₅₀ of the IATS 13isolate and five of five mice challenged with the IATS 6 isolate. Thecell line 8H7 was deposited with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852, on Nov. 4, 1986 and assignedaccession number ATCC No. CRL 9258.

From the foregoing, it will be appreciated that the cell lines of thepresent invention provide human monoclonal antibodies and fragmentsthereof cross-reactive for and cross-protective against various P.aeruginosa IATS serotypes. This allows prophylactic and therapeuticcompositions to be more easily developed that can be effective againstinfections due to most, if not all, P. aeruginosa strains. In addition,the cell lines provide antibodies which find uses in immunoassays andother well-known procedures.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

We claim:
 1. A composition comprising a human monoclonal antibody orbinding fragment thereof which specifically binds to accessiblelipopolysaccharide determinants of IATS serotypes 2 and 5 and Fisherimmunotype 3 of Pseudomonas aeruginosa, wherein said monoclonal antibodyor binding fragment thereof inhibits the viability of at least two ofsaid IATS serotypes and immunotype in the presence of the complement andneutrophils.
 2. The composition of claim 1 wherein the monoclonalantibody or binding fragment thereof further binds to and inhibits theviability of P. aeruginosa organisms of Fisher immunotype
 7. 3. Thecomposition of claim 1, wherein the monoclonal antibody or bindingfragment thereof has the antigen binding specificity of a monoclonalantibody or binding fragment thereof obtained from ATCC CRL
 8941. 4. Thecomposition of claim 3, wherein the monoclonal antibody or bindingfragment thereof is obtained from ATCC CRL
 8941. 5. A compositioncomprising a human monoclonal antibody or binding fragment thereof whichspecifically binds to accessible lipopolysaccharide determinants ofPseudomonas aeruginosa, wherein said monoclonal antibody or bindingfragment thereof are from ATCC CRL
 9258. 6. A composition comprising ahuman monoclonal antibody or binding fragment thereof which specificallybinds to accessible outer membrane lipopolysaccharide determinants ofPseudomonas aeruginosa, wherein said monoclonal antibody or bindingfragment thereof are obtained from ATCC CRL 9171.